In general, amphipathic molecules have distinct regions of hydrophilic character and distinct regions of hydrophobic character. Amphipathic molecules form three types of macromolecular structures when dispersed in water: micelles, hexagonal phase and lipid bilayers. The exact macromolecular structure depends in part, to the relative sizes of the hydrophilic and hydrophobic regions of the molecule.
In certain instances, when the cross-sectional area of the hydrophilic region of the molecule is slightly less than, or equal to, that of the hydrophobic part of the molecule, such as in many phospholipids, the formation of bilayers is favored. Phospholipids contain one phosphate, a glycerol and one or more fatty acids. These molecules form lipid bilayers that are two-dimensional sheets in which all of the hydrophobic portions, e.g., acyl side chains, are shielded from interaction with water except those at the ends of the sheet. These bilayers form three-dimensional vesicles known as liposomes.
Liposomes are self-assembling structures comprising one or more bilayers of amphipathic lipid molecules that enclose an internal aqueous volume. The amphipathic lipid molecules that make up the lipid bilayers comprise a polar headgroup region covalently linked to one or two non-polar acyl chains. In certain instances, the energetically unfavorable contact between the hydrophobic acyl chains and the aqueous solution surrounding the lipid molecules causes them to rearrange and thus, the polar headgroups are oriented towards the aqueous solution, while the acyl chains orient towards the interior part of the bilayer. The lipid bilayer structure comprises two opposing monolayers, wherein the acyl chains are shielded from coming into contact with the surrounding medium.
Liposomes are excellent vehicles for drug delivery. In a liposome-drug delivery system, an active ingredient, such as a drug, is encapsulated or entrapped in the liposome and then administered to the patient to be treated. Alternatively, if the active ingredient is lipophilic, it may associate with the lipid bilayer. Active ingredients entrapped within liposomes can reduce toxicity, increase efficacy, or both.
One area of liposome research has been the design of the "trigger" for the liposome to release its payload or active agent. Various parameters to initiate release can be used, which include pH, ionic strength, controlled release and antibody attachment. Past developments of pH-sensitive liposomes have focused principally in the area of anionic liposomes comprised largely of phosphatidylethanolamine (PE) bilayers (see, Huang et al., Biochemistry, 28, 9508-9514 (1989); Duzgunes et al., pH-Sensitive Liposomes, in Membrane Fusion 1990, pp. 713-730; Wilschut, J. and Hoekstra, D. (eds.), Marcel-Decker Inc., New York. and Yatvin et al., Science, 210, 1253-1255 (1980)). The addition of lipids containing carboxylate groups (e.g., hemisuccinate, oleic acid, etc.) to PE lipids help stabilize bilayer morphology at nonacidic pH. After cellular internalization occurs via endocytosis of PE liposome preparations containing carboxylate lipids, endosomal acidification progresses. As the acidification progresses below the pK of the carboxylic acid lipid, the carboxylate groups are gradually neutralized and the PE-rich bilayer is destabilized.
PE lipids are prone to assume the inverted hexagonal phase (H.sub.II) in the absence of stabilizing influences such as the presence of negatively charged head groups. In this instance, the hydrophobic region of the molecule is greater than that of the hydrophilic part of the molecule. Thus, lipids are released from liposome aggregates as the pH is lowered, resulting in extensive mixing with the endosomal membrane and improved cytoplasmic delivery of the liposome contents.
More recently, pH-sensitive cationic liposomes have been developed to mediate transfer of DNA into cells. For instance, researchers described a series of amphiphiles with headgroups containing imidazole, methylimidazole, or aminopyridine moieties (see, Budker et al., Nature Biotech., 14, 760-764 (1996)). The amine-based headgroups possess pKs within the physiologic range of between 4.5 to 8. The hydrophobic domains for these lipids varied and included cholesterol and dioleoyl or dipalmitoyl glycerol. The pH sensitivity is a result of their titratable amine headgroups, a feature previously exploited that demonstrated pH-dependent fusion of liposomes containing poly-L-histidine (see, Uster et al., Biochemistry, 24, 1-8 (1985)). Acidification results in headgroup protonation and increases the effective headgroup size via electropositive repulsions. It has been postulated that the increased positive charge also increases the interactions with DNA and the anionic components of the endosomal membrane. These pH-dependent changes are believed to disrupt the liposome integrity, thus leading to fusion with the endosomal membrane and greater DNA escape. Thus, in the prior art methods, a decrease in pH causes assembly (e.g., liposome) reorganization.
In view of the foregoing, and in contrast to the known methods for reorganizing lipid assemblies as a function of pH, what is needed in the art are lipid molecules within the assemblies that are capable of structural reorganization upon a change in pH. Methods that use these lipids in liposome formulations in nucleic acid transfection are also needed. The present invention fulfills these as well as other needs.